This grant proposal seeks to develop candidate vaccines against Ebola virus using a unique noncytopathic replicon RNA-based vaccine system derived from the Australian flavivirus Kunjin (KUN) and to evaluate their ability to induce efficient and long-term immune responses in mice and protection against Ebola virus challenge in cynomolgus macaques. The main feature of the system is the ability of KUN replicon RNA to continuously support its own replication in cell cytoplasm without harming the host cell which results in sustained enhanced level of immunogen expression. The functional replicon RNAs encoding immunogenes will be delivered by two modalities: (i) as virus-like particles (VLPs) containing replicon RNA packaged into the KUN envelope, and (ii) as plasmid DNA containing KUN replicon cDNA placed under the control of a mammalian expression promoter. Individual Ebola virus proteins GP, NP, VP24, VP40, and VP40-GP cassettee will be cloned into the appropriate KUN replicon vectors and expression of Ebola proteins will first be examined in vitro by western blot and radioimmunoprecipitation analyses. The immunogenicity of the VLP-based and DNA-based KUN-Ebola vaccines will be initially tested in mice. Induction of both antibody and CD8+ T cell responses will be examined and compared with that induced by immunization with conventional plasmid DNA vaccines expressing the same Ebola genes. The final stage of the project will be the evaluation of protective properties of the KUN-Ebola replicon vaccines in cynomolgous macaques against challenge with Ebola virus Zair strain. KUN replicon vaccine vectors produce high level of immunogen for extended periods without inducing cytopathic effects. We postulate that these features of KUN replicon vectors will allow efficient induction and maintenance of antibodies and effector CD8+ T cells, a feature likely to be beneficial for protection against rapidly replicating viruses like Ebola.